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Understanding cellular processes can provide insight into disease pathogenesis and reveal critical information for prevention, diagnosis, and treatment. As key executors and signaling regulators, proteins carry relevant information not available from genomics and transcriptomics. Cell-to-cell differences significantly affect disease incidence and drug responses, generating a need for protein analysis at

Understanding cellular processes can provide insight into disease pathogenesis and reveal critical information for prevention, diagnosis, and treatment. As key executors and signaling regulators, proteins carry relevant information not available from genomics and transcriptomics. Cell-to-cell differences significantly affect disease incidence and drug responses, generating a need for protein analysis at the single-cell level. However, quantitative protein analysis at the single-cell level remains challenging due to the low protein amount in a single cell and the proteome complexity. It requires sensitive detection techniques and appropriate sample preparation and delivery to the detection area. Here, a microfluidic platform in tandem with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been developed for targeted intracellular protein analysis. The elastomeric multi-layer microfluidic platform, termed MIMAS, was designed as a series of 8.75 nL wells separated by pneumatic valves. The MIMAS platform allows cell loading, sample processing on-chip, and further in situ mass spectrometry analysis. The sample processing includes cell lysis, immunocapture, tryptic digestion and MALDI matrix solution loading for co-crystallization. This work demonstrates that the MIMAS approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 from MCF-7 breast cancer cells using an isotope-labeled peptide. The limit of detection was determined as 11.22 nM, equivalent to 5.91 x 10^7 protein molecules per well. Moreover, the MIMAS platform design was improved, allowing the successful quantification of Bcl-2 protein in small cell ensembles down to ~10 cells in 4 nL wells. Furthermore, the MIMAS platform was integrated with laser capture microdissection (LCM) for protein analysis from post-mortem human tissues. Intracellular amyloid-β peptide (Aβ), a hallmark of Alzheimer’s Disease, was assessed from human brain tissue using the LCM-MIMAS. The successful detection of Aβ from small cell ensembles (20 sliced pyramidal cells) demonstrated the LCM-MIMAS capability of assessing intracellular proteins from specific tissue cell subpopulations. The MIMAS approach is a promising tool for intracellular protein analysis from small cell ensembles, with the potential for single-cell analysis. It allows for protein analysis towards the understanding of biological phenomena for clinical and biological research.
ContributorsCruz Villarreal, Jorvani (Author) / Ros, Alexandra (Thesis advisor) / Borges, Chad R (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2022
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Description
The imaging and detection of specific cell types deep in biological tissue is critical for the diagnosis of cancer and the study of biological phenomena. Current high-resolution optical imaging techniques are depth limited due to the high degree of optical scattering that occurs in tissues. To address these limitations, photoacoustic

The imaging and detection of specific cell types deep in biological tissue is critical for the diagnosis of cancer and the study of biological phenomena. Current high-resolution optical imaging techniques are depth limited due to the high degree of optical scattering that occurs in tissues. To address these limitations, photoacoustic (PA) techniques have emerged as noninvasive methods for the imaging and detection of specific biological structures at extended depths in vivo. In addition, near-infrared (NIR) contrast agents have further increased the depth at which PA imaging can be achieved in biological tissues. The goal of this research is to combine novel PA imaging and NIR labeling strategies for the diagnosis of disease and for the detection of neuronal subtypes. Central Hypothesis: Utilizing custom-designed PA systems and NIR labeling techniques will enable the detection of specific cell types in vitro and in mammalian brain slices. Work presented in this dissertation addresses the following: (Chapter 2): The custom photoacoustic flow cytometry system combined with NIR absorbing copper sulfide nanoparticles for the detection of ovarian circulating tumor cells (CTCs) at physiologically relevant concentrations. Results obtained from this Chapter provide a unique tool for the future detection of ovarian CTCs in patient samples at the point of care. (Chapter 3): The custom photoacoustic microscopy (PAM) system can detect genetically encoded near-infrared fluorescent proteins (iRFPs) in cells in vitro. Results obtained from this Chapter can significantly increase the depth at which neurons and cellular processes can be targeted and imaged in vitro. (Chapter 4): Utilizing the Cre/lox recombination system with AAV vectors will enable selective tagging of dopaminergic neurons with iRFP for detection in brain slices using PAM. Thus, providing a new means of increasing the depth at which neuronal subtypes can be imaged and detected in the mammalian brain. Significance: Knowledge gained from this research could have significant impacts on the PA detection of ovarian cancer and extend the depth at which neuronal subtypes are imaged in the mammalian brain.
ContributorsLusk, Joel F. (Author) / Smith, Barbara S. (Thesis advisor) / Halden, Rolf (Committee member) / Anderson, Trent (Committee member) / Arizona State University (Publisher)
Created2022
Description

Advancing the understanding and treatment of many neurological disorders can be achieved by improving methods of neuronal detection at increased depth in the mammalian brain. Different cell subtypes cannot be detected using non-invasive techniques beyond 1 mm from cortical surface, in the context of targeting particular cell types in vivo

Advancing the understanding and treatment of many neurological disorders can be achieved by improving methods of neuronal detection at increased depth in the mammalian brain. Different cell subtypes cannot be detected using non-invasive techniques beyond 1 mm from cortical surface, in the context of targeting particular cell types in vivo (Wang, 2012). These limitations in the depth of imaging and targeting are due to optical scattering (Ntziachristos, 2010). In order to overcome these restrictions, longer wavelength fluorescent proteins have been utilized by researchers to see tagged cells at depth. Optical techniques such as two-photon and confocal microscopy have been used in combination with fluorescent proteins to expand depth, but are still limited by the penetration depth of light due to optical scattering (Lee, 2015). This research aims to build on other detection methods, such as the photoacoustic effect and automated fluorescence-guided electrophysiology, to overcome this limitation.

ContributorsAridi, Christina (Author) / Smith, Barbara (Thesis director) / Marschall, Ethan (Committee member) / Barrett, The Honors College (Contributor) / Watts College of Public Service & Community Solut (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
Description

Polymeric nanoparticles (NP) consisting of Poly Lactic-co-lactic acid - methyl polyethylene glycol (PLLA-mPEG) or Poly Lactic-co-Glycolic Acid (PLGA) are an emerging field of study for therapeutic and diagnostic applications. NPs have a variety of tunable physical characteristics like size, morphology, and surface topography. They can be loaded with therapeutic and/or

Polymeric nanoparticles (NP) consisting of Poly Lactic-co-lactic acid - methyl polyethylene glycol (PLLA-mPEG) or Poly Lactic-co-Glycolic Acid (PLGA) are an emerging field of study for therapeutic and diagnostic applications. NPs have a variety of tunable physical characteristics like size, morphology, and surface topography. They can be loaded with therapeutic and/or diagnostic agents, either on the surface or within the core. NP size is an important characteristic as it directly impacts clearance and where the particles can travel and bind in the body. To that end, the typical target size for NPs is 30-200 nm for the majority of applications. Fabricating NPs using the typical techniques such as drop emulsion, microfluidics, or traditional nanoprecipitation can be expensive and may not yield the appropriate particle size. Therefore, a need has emerged for low-cost fabrication methods that allow customization of NP physical characteristics with high reproducibility. In this study we manufactured a low-cost (<$210), open-source syringe pump that can be used in nanoprecipitation. A design of experiments was utilized to find the relationship between the independent variables: polymer concentration (mg/mL), agitation rate of aqueous solution (rpm), and injection rate of the polymer solution (mL/min) and the dependent variables: size (nm), zeta potential, and polydispersity index (PDI). The quarter factorial design consisted of 4 experiments, each of which was manufactured in batches of three. Each sample of each batch was measured three times via dynamic light scattering. The particles were made with PLLA-mPEG dissolved in a 50% dichloromethane and 50% acetone solution. The polymer solution was dispensed into the aqueous solution containing 0.3% polyvinyl alcohol (PVA). Data suggests that none of the factors had a statistically significant effect on NP size. However, all interactions and relationships showed that there was a negative correlation between the above defined input parameters and the NP size. The NP sizes ranged from 276.144 ± 14.710 nm at the largest to 185.611 ± 15.634 nm at the smallest. In conclusion, the low-cost syringe pump nanoprecipitation method can achieve small sizes like the ones reported with drop emulsion or microfluidics. While there are trends suggesting predictable tuning of physical characteristics, significant control over the customization has not yet been achieved.

ContributorsDalal, Dhrasti (Author) / Stabenfeldt, Sarah (Thesis director) / Wang, Kuei-Chun (Committee member) / Flores-Prieto, David (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
Description

The purpose of this research thesis paper is to provide further insight into the development of extended reality (XR), augmented reality (AR), and virtual reality (VR) technologies within the educational space and survey how well they are received as well as whether or not they can provide additional learning benefit

The purpose of this research thesis paper is to provide further insight into the development of extended reality (XR), augmented reality (AR), and virtual reality (VR) technologies within the educational space and survey how well they are received as well as whether or not they can provide additional learning benefit in regards to other learning mediums such as reading textbooks, watching videos on the subject matter, and other such more traditional mediums. The research conducted consisted of a collaborative effort alongside the School of Biological and Health Systems Engineering (SBHSE) personnel and using their provided resources in order to generate a framework with the aforementioned technology, to aid in the development of a web-based XR system which will serve primarily as a means for SBHSE students at Arizona State University (ASU) to enhance their learning experience when it comes to topics such as anatomy and physiology of the human body, with the potential of extending this technology towards other subject matters as well, such as other STEM-related fields. Information about the initial research which included an analysis of the pertinent readings that support a benefit to using XR technology as a means to deliver course content is what is first focused on throughout this document. Then, the process that went into the design and development of the base framework that was in joint collaboration with the SBHSE will be covered. And, to conclude, a case study to generate applicable data to support the argument is covered as well as the results from it, which presented a potential for a future development plan and next steps plan once the developed materials and research are handed off.

ContributorsMihaylov, Dimitri (Author) / Chavez-Echeagaray, Maria Elena (Thesis director) / Farzam, Maziar (Committee member) / Barrett, The Honors College (Contributor) / Dean, W.P. Carey School of Business (Contributor) / Computer Science and Engineering Program (Contributor)
Created2023-05
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Description
Characterizing and identifying neuroinflammatory states is crucial in developing treatments for neurodegenerative diseases. Microglia, the resident immune cells of the brain, regulate inflammation and play a vital role in maintaining brain health by producing cytokines, performing phagocytosis, and inducing or reducing inflammation. These functional states can be described by specific

Characterizing and identifying neuroinflammatory states is crucial in developing treatments for neurodegenerative diseases. Microglia, the resident immune cells of the brain, regulate inflammation and play a vital role in maintaining brain health by producing cytokines, performing phagocytosis, and inducing or reducing inflammation. These functional states can be described by specific patterns of gene expression called transcriptional programs, which are determined by the activity of a set of key transcription factors that have mostly been identified. Thus, an assay for transcription factor activity could reveal the state of the microglial cells and neuroinflammation across the brain. This research developed an assay that uses a transcription factor dependent reporter to indicate which transcriptional programs are activated in the cell when exposed to different stimuli. The prototype assay quantifies nuclear factor kappa B (NF-kB) response in cultured human cells. NF-kB is a well-characterized transcription factor associated with inflammatory pathways in most cells, including microglia. The reporter construct contains an NF-kB specific responsive element that can induce fluorescence/luminescence upon activation of the transcription factor. In an iterative refinement, a dual response fluorescent reporter was developed, which uses a secondary constitutively fluorescent reporter for built-in normalization of the responsive element for microscopy studies. With further refinement, this modular system will serve as a template for less understood transcriptional enhancers allowing for rapid, low-cost assays of neuroimmune regulators and potential in vivo applications in the study of neuroinflammation.
ContributorsLieberman, Emma (Author) / Bartelle, Benjamin B (Thesis advisor) / Plaisier, Christopher L (Committee member) / Andrews, Madeline G (Committee member) / Arizona State University (Publisher)
Created2023
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Description
Chimeric antigen receptor (CAR)-T cell therapy is a type of cancer immunotherapy has shown promising results in engineering the T cells which targets a specific antigen. Despite their success rate, there are certain limitations to the use of CAR-T therapies that includes cytokine release syndrome (CRS), neurologic toxicity, lack of

Chimeric antigen receptor (CAR)-T cell therapy is a type of cancer immunotherapy has shown promising results in engineering the T cells which targets a specific antigen. Despite their success rate, there are certain limitations to the use of CAR-T therapies that includes cytokine release syndrome (CRS), neurologic toxicity, lack of response in approximately 50% of treated patients, monitoring of patients treated with CAR-T therapy. However, rapid point- of- care testing helps in quantifying the circulating CAR T cells and can enhance the safety of patients, minimize the cost of CAR-T cell therapy, and ease the management process. Currently, the standard method to quantify CAR-T cell in patient blood samples are flow cytometry and quantitative polymerase chain reaction (qPCR). But these techniques are expensive and are not easily accessible and suitable for point- of- care testing to assist real- time clinical decisions. To overcome these hurdles, here I propose a solution to these problems by rapid optical imaging (ROI)- based principle to monitor and detect CAR-T cells. In this project, a microfluidic device is developed and integrated with two functions: (1) Centrifuge free, filter- based separation of white blood cells and plasma; (2) Optical imaging- based technique for digital counting of CAR T- cells. Here, I carried out proof- of- concept test on the laser cut prototype microfluidic chips as well as the surface chemistry for specific capture of CAR-T cells. These data show that the microfluidic chip can specifically capture CAR-T positive cells with concentration dependent counts of captured cells. Further development of the technology could lead to a new tool to monitor the CAR-T cells and help the clinicians to effectively measure the efficacy of CAR-T therapy treatment in a faster and safer manner.
ContributorsElanghovan, Praveena (Author) / Wang, Shaopeng (Thesis advisor) / Forzani, Erica (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2023
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Description
Placental pregnancy is a biological scenario where tissue types bearing different antigen signatures co-exist within the same microenvironment without rejection. Placental trophoblast cells locally modulate the immune system in pregnancy, and one process through which this occurs is through the release of a class of nano-scaled extracellular vesicles called exosomes.

Placental pregnancy is a biological scenario where tissue types bearing different antigen signatures co-exist within the same microenvironment without rejection. Placental trophoblast cells locally modulate the immune system in pregnancy, and one process through which this occurs is through the release of a class of nano-scaled extracellular vesicles called exosomes. The aim is to use these placental-derived immunomodulatory exosomes as a therapeutic and engineer a means to deliver these exosomes using a hydrogel vehicle. As such, two representative trophoblast cell lines, JAR and JEG-3, were used as exosome sources. First step involved the evaluation of the morphological and proteomic characterization of the isolated exosomes through dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM) imaging, and mass spectrometry (MS) analysis. Following exosome characterization, incorporation of exosomes within hydrogel matrices like polyethylene glycol and alginate to determine their release profile over a timescale of 14 days was performed. Comparing the release between the two cell lines isolated exosomes, no discernible difference is observed in their release, and release appears complete within two days. Future studies will evaluate the impact of exosome loadings and hydrogel modification on exosome release profiles, as well as their influence on immune cells.
ContributorsHiremath, Shivani Chandrashekher Swamy (Author) / Weaver, Jessica D (Thesis advisor) / Plaisier, Christopher (Committee member) / Wang, Kuei-Chun (Committee member) / Arizona State University (Publisher)
Created2021
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Description
While pulse oximeter technology is not necessarily an area of new technology, advancements in performance and package of pulse sensors have been opening up the opportunities to use these sensors in locations other than the traditional finger monitoring location. This research report examines the full potential of creating a

While pulse oximeter technology is not necessarily an area of new technology, advancements in performance and package of pulse sensors have been opening up the opportunities to use these sensors in locations other than the traditional finger monitoring location. This research report examines the full potential of creating a minimally invasive physiological and environmental observance method from the ear location. With the use of a pulse oximeter and accelerometer located within the ear, there is the opportunity to provide a more in-depth means to monitor a pilot for a Gravity-Induced Loss of Consciousness (GLOC) scenario while not adding any new restriction to the pilot's movement while in flight. Additionally, building from the GLOC scenario system, other safety monitoring systems for military and first responders are explored by alternating the physiological and environmental sensors. This work presents the design and development of hardware, signal processing algorithms, prototype development, and testing results of an in-ear wearable physiological sensor.
ContributorsNichols, Kevin (Author) / Redkar, Sangram (Thesis advisor) / Tripp Jr., Llyod (Committee member) / Dwivedi, Prabha (Committee member) / Sugar, Thomas (Committee member) / Arizona State University (Publisher)
Created2021
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Description
Sutures, staples, and tissue glues remain the primary means of tissue approximation and vessel ligation. Laser-activated tissue sealing is an alternative approach that conventionally employs light-absorbing chromophores and nanoparticles for converting near-infrared (NIR) laser to heat. The local increase in temperature engenders interdigitation of sealant and tissue biomolecules, resulting in

Sutures, staples, and tissue glues remain the primary means of tissue approximation and vessel ligation. Laser-activated tissue sealing is an alternative approach that conventionally employs light-absorbing chromophores and nanoparticles for converting near-infrared (NIR) laser to heat. The local increase in temperature engenders interdigitation of sealant and tissue biomolecules, resulting in rapid tissue sealing. Light-activated sealants (LASE) were developed in which indocyanine green (ICG) dye is embedded within a biopolymer matrix (silk or chitosan) for incisional defect repair. Light-activated tissue-integrating sutures (LATIS) that synergize the benefits of conventional suturing and laser sealing were also fabricated and demonstrated higher efficacies for tissue biomechanical recovery and repair in a full-thickness, dorsal surgical incision model in mice compared to commercial sutures and cyanoacrylate skin glue. Localized delivery of modulators of tissue repair, including histamine and copper, from LASE and LATIS further improved healed skin strength. In addition to incisional wounds, histamine co-delivered with silk fibroin LASE films accelerated the closure of full thickness, splinted excisional wounds in immunocompetent BALB/c mice and genetically obese and diabetic db/db mice, resulting in faster closure than Tegaderm wound dressing. Immunohistochemistry analyses showed LASE-histamine treatment enhanced wound repair involving mechanisms of neoangiogenesis, myofibroblast activation, transient epidermal EMT, and also improve healed skin biomechanical strength which are hallmarks of improved healing outcomes. Benefit of temporal delivery was further investigated of a second therapeutic (growth factor nanoparticles) in modulating wound healing outcomes in both acute and diabetic wounds. The hypothesis of temporal delivery of second therapeutic around the ‘transition period’ in wounds further improved wound closure kinetics and biomechanical recovery of skin strength. Laser sealing and approximation, together with delivery of immunomodulatory mediators, can lead to faster healing and tissue repair, thus reducing wound dehiscence, preventing wounds moving towards chronicity and lowering incidence of surgical site infections, all of which can have significant impact in the clinic.
ContributorsGhosh, Deepanjan (Author) / Rege, Kaushal (Thesis advisor) / Acharya, Abhinav (Committee member) / Holloway, Julianne (Committee member) / DiCaudo, David (Committee member) / P. Leung, Kai (Committee member) / Arizona State University (Publisher)
Created2021